Molecular Biology of A

In order to avoid toxicity to normal cells, replication-competent adenoviruses have been designed to be tumor selective. Although targeting (selectivity) can be achieved by a variety of approaches (30), in the context of adenoviruses, tumor selectivity has been most successfully accomplished by altering the viral genes that are required for viral replication in normal cells but are dispensable in cancer cells (31,32). This approach was first pursued by Bischoff et al., who developed a conditionally replicative adenovirus, ONYX-015, that contains a deletion in the viral p53-binding protein E1B-55kDa and

Fig. 2. Adenoviruses as oncolytic agents. Infection of a few cells by replication-competent adenoviruses, such as A 24, results in amplification of the virus with subsequent release and infection of surrounding cells. Each round of infection and lysis not only kills the infected cells but also increases the number of viral particles. Selectivity for tumor cells is determined by genetically engineering the virus. (Reproduced with permission from ref. 64.)

Fig. 2. Adenoviruses as oncolytic agents. Infection of a few cells by replication-competent adenoviruses, such as A 24, results in amplification of the virus with subsequent release and infection of surrounding cells. Each round of infection and lysis not only kills the infected cells but also increases the number of viral particles. Selectivity for tumor cells is determined by genetically engineering the virus. (Reproduced with permission from ref. 64.)

that was intended to selectively replicate in tumor cells containing an inactivated p53 protein (31). Fueyo et al. also used this approach to develop A 24, whose selectivity is based on a deletion in the E1A viral protein and which, therefore, selectively replicates in tumor cells containing inactivated retinoblastoma (Rb) protein (17). To understand the basis of the selectivity of A 24 for tumor cells vs normal cells, the roles of cellular Rb protein and viral E1A protein in cell cycle regulation and viral replication must be understood (see Fig. 3).

3.1.1. Regulation of the Cell Cycle: The Rb Protein

The control of cell division is a complex process (for review see 33,34), but to a first approximation, the critical regulator of entry from G1 to S-phase is the retinoblastoma (Rb) protein (35), a 110-kDa phosphoprotein whose phosphorylation status regulates the activity of E2F, a transcription factor that is important in activating genes involved in DNA replication (S-phase) and other components of cell proliferation (35-39). Hypophosphorylated Rb binds E2F, and the resulting E2F/Rb complex represses the expression of E2F-target genes, thereby arresting cells in G1. Phosphorylation of Rb diminishes the repression of E2F by Rb, resulting in transcriptional activation of E2F-target genes and progression into S-phase (see Fig. 3). Phosphorylation of Rb is accomplished through cyclin-dependent kinases (in particular, cdks 4 and 6), a group of serine/threonine kinases whose activity requires association with regulatory subunits or

Fig. 3. The basis of tumor selectivity for A 24. Wild-type adenovirus commandeers the cellular replication machinery by inactivating Rb protein by binding the E1A protein (top). This interaction frees up E2F, thereby driving the cell into S-phase and promoting viral replication. Because it harbors a mutant E1A protein, A 24 is unable to inactivate Rb (bottom left) and thus cannot replicate in normal cells. Nevertheless, tumor cells typically contain an inactivated Rb pathway, rendering the mutant E1A of Delta 24 unnecessary for promoting cell cycle progression, and viral replication proceeds unhindered.

Fig. 3. The basis of tumor selectivity for A 24. Wild-type adenovirus commandeers the cellular replication machinery by inactivating Rb protein by binding the E1A protein (top). This interaction frees up E2F, thereby driving the cell into S-phase and promoting viral replication. Because it harbors a mutant E1A protein, A 24 is unable to inactivate Rb (bottom left) and thus cannot replicate in normal cells. Nevertheless, tumor cells typically contain an inactivated Rb pathway, rendering the mutant E1A of Delta 24 unnecessary for promoting cell cycle progression, and viral replication proceeds unhindered.

cyclins (cyclin D1 is most critical in the Rb pathway). Cdks and their inhibitors (CDKIs, particularly p16/INK4a and p21) are important checkpoint proteins in regulating Rb and thus cell-cycle progression (40).

3.1.2. Inactivation of Rb in Tumor Cells

Evidence is accumulating that nearly all tumors, including gliomas, harbor some defect in Rb or its regulatory pathway (40,41). Although mutational inactivation of Rb is less common in gliomas than in other cancers, loss of p16INK4a (CDK-inhibitor upstream of Rb) or overexpression of cdks 4 and 6 or cyclin D are common defects in gliomas (42). This loss of Rb function in tumor cells results in high levels of E2F, causing unregulated entry into the cell cycle and unfettered cellular proliferation, a cardinal feature of cancer (43) (see Fig. 3).

3.1.3. Forced Entry by Adenoviruses into the Cell Cycle: E1A Inactivation of Rb

Inactivation of Rb is also fundamental to the replication of most viruses, including adenoviruses (44,45). After entry into cells, viruses replicate their DNA and produce the proteins of the structural capsid before being released by cell lysis. Because adenoviruses contain a small genome that encodes only about 30 different mRNA transcripts, replication relies upon commandeering cellular replication machinery. It is the role of the so-called early genes (E1-4) to prepare infected cells for viral DNA replication. Cellular entry into S-phase is regulated by viral E1A proteins (see Fig. 3). E1A is the first viral gene transcribed after infection, and its expression produces two related proteins, 243R and 289R. The 289R protein binds to and inactivates cellular Rb. ElA-mediated sequestration of Rb de-represses the transcriptional properties of E2F and allows for entry into S-phase and replication of the viral genome.

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